ATP-consuming futile cycles as energy dissipating mechanisms to counteract obesity

Obesity results from an imbalance in energy homeostasis, whereby excessive energy intake exceeds caloric expenditure. Energy can be dissipated out of an organism by producing heat (thermogenesis), explaining the long-standing interest in exploiting thermogenic processes to counteract obesity. Mitochondrial uncoupling is a process that expends energy by oxidizing nutrients to produce heat, instead of ATP synthesis. Energy can also be dissipated through mechanisms that do not involve mitochondrial uncoupling. Such mechanisms include futile cycles described as metabolic reactions that consume ATP to produce a product from a substrate but then converting the product back into the original substrate, releasing the energy as heat. Energy dissipation driven by cellular ATP demand can be regulated by adjusting the speed and number of futile cycles. Energy consuming futile cycles that are reviewed here are lipolysis/fatty acid re-esterification cycle, creatine/phosphocreatine cycle, and the SERCA-mediated calcium import and export cycle. Their reliance on ATP emphasizes that mitochondrial oxidative function coupled to ATP synthesis, and not just uncoupling, can play a role in thermogenic energy dissipation. Here, we review ATP consuming futile cycles, the evidence for their function in humans, and their potential employment as a strategy to dissipate energy and counteract obesity.

Retroactivity induced operating regime transition in an enzymatic futile cycle

Activated phosphorylation-dephosphorylation biochemical reaction cycles are a class of enzymatic futile cycles. A futile cycle such as a single MAPK cascade governed by two underlying enzymatic reactions permits Hyperbolic (H), Signal transducing (ST), Threshold-hyperbolic (TH) and Ultrasensitive (U) operating regimes that characterize input-output behaviour. Retroactive signalling caused by load due to sequestration of phosphorylated or unphosphorylated form of the substrate in a single enzymatic cascade without explicit feedback can introduce two-way communication, a feature not possible otherwise. We systematically characterize the operating regimes of a futile cycle subject to retroactivity in either of the substrate forms. We demonstrate that increasing retroactivity strength, which quantifies the downstream load, can trigger five possible regime transitions. Retroactivity strength is a reflection of the fraction of the substrate sequestered by its downstream target. Remarkably, the minimum required retroactivity strength to evidence any sequestration triggered regime transition demands 23% of the substrate bound to its downstream target. This minimum retroactivity strength corresponds to the transition of the dose-response curve from ST to H regime. We show that modulation of the saturation and unsaturation levels of the enzymatic reactions by retroactivity is the fundamental mechanism governing operating regime transition.

Impact of Altered Trehalose Metabolism on Physiological Response of Penicillium chrysogenum Chemostat Cultures during Industrially Relevant Rapid Feast/Famine Conditions

Due to insufficient mass transfer and mixing issues, cells in the industrial-scale bioreactor are often forced to experience glucose feast/famine cycles, mostly resulting in reduced commercial metrics (titer, yield and productivity). Trehalose cycling has been confirmed as a double-edged sword in the Penicillium chrysogenum strain, which facilitates the maintenance of a metabolically balanced state, but it consumes extra amounts of the ATP responsible for the repeated breakdown and formation of trehalose molecules in response to extracellular glucose perturbations. This loss of ATP would be in competition with the high ATP-demanding penicillin biosynthesis. In this work, the role of trehalose metabolism was further explored under industrially relevant conditions by cultivating a high-yielding Penicillium chrysogenum strain, and the derived trehalose-null strains in the glucose-limited chemostat system where the glucose feast/famine condition was imposed. This dynamic feast/famine regime with a block-wise feed/no feed regime (36 s on, 324 s off) allows one to generate repetitive cycles of moderate changes in glucose availability. The results obtained using quantitative metabolomics and stoichiometric analysis revealed that the intact trehalose metabolism is vitally important for maintaining penicillin production capacity in the Penicillium chrysogenum strain under both steady state and dynamic conditions. Additionally, cells lacking such a key metabolic regulator would become more sensitive to industrially relevant conditions, and are more able to sustain metabolic rearrangements, which manifests in the shrinkage of the central metabolite pool size and the formation of ATP-consuming futile cycles.

Nucleotide-mediated allosteric regulation of bifunctional Rel enzymes

Bifunctional Rel stringent factors, the most broadly distributed class of RSHs, are ribosome-associated enzymes that transfer a pyrophosphate group from ATP onto the 3′ of GTP or GDP to synthesize (p)ppGpp and also catalyse the 3′ pyrophosphate hydrolysis of the alarmone to degrade it. The precise regulation of these enzymes seems to be a complex allosteric mechanism, and despite decades of research, it is unclear how the two opposing activities of Rel are controlled at the molecular level. Here we show that a stretch/recoil guanosine-switch mechanism controls the catalytic cycle of T. thermophilus Rel (RelTf). The binding of GDP/ATP stretches apart the NTD catalytic domains of RelTf (RelTtNTD) activating the synthetase domain and allosterically blocking the hydrolase active site. Conversely, binding of ppGpp unlocks the hydrolase domain and triggers recoil of both NTDs, which partially buries the synthetase active site and precludes the binding of synthesis precursors. This allosteric mechanism acts as an activity switch preventing futile cycles of alarmone synthesis and degradation.

Why is cyanide acutely lethal at very low doses?

Cyanide is conventionally perceived as a binder of heme-Fe centers, disrupting oxygen transport by blood hemoglobin and mitochondrial cytochrome oxidase function. This explanation of toxicity would require millimolar (g/Kg dosage) concentration of cyanide, whereas it is lethal even at micromolar (mg/Kg dosage) ranges. It is long known that oxygen consumption by cells leads to the production of diffusible reactive oxygen species (DROS). Recently, DROS mediated catalytic/metabolic roles were proposed as a physiological source of heat and phosphorylation of ADP within mitochondria. In this purview, it is hypothesized herein that cyanide uses the catalytic DROS via futile cycles, stopping ATP-synthesis and thus killing cells. A quantitative mechanistic perspective delineating the old and new explanations is provided herein. Further, experimental modalities and predictable outcomes are detailed to test the new hypothesis.

Ubiquitin-dependent folding of the Wnt signaling coreceptor LRP6

Many membrane proteins fold inefficiently and require the help of enzymes and chaperones. Here we reveal a novel folding assistance system that operates on membrane proteins from the cytosolic side of the endoplasmic reticulum (ER). We show that folding of the Wnt signaling coreceptor LRP6 is promoted by ubiquitination of a specific lysine, retaining it in the ER while avoiding degradation. Subsequent ER exit requires removal of ubiquitin from this lysine by the deubiquitinating enzyme USP19. This ubiquitination-deubiquitination is conceptually reminiscent of the glucosylation-deglucosylation occurring in the ER lumen during the calnexin/calreticulin folding cycle. To avoid infinite futile cycles, folded LRP6 molecules undergo palmitoylation and ER export, while unsuccessfully folded proteins are, with time, polyubiquitinated on other lysines and targeted to degradation. This ubiquitin-dependent folding system also controls the proteostasis of other membrane proteins as CFTR and anthrax toxin receptor 2, two poor folders involved in severe human diseases.